This project integrates DNA microarrays, bioinformatics and epidemiology to identify signature patterns of gene expression in head and neck squamous cell carcinomas (HNSSCs), to advance understanding of oncogenesis and to discover novel biomarkers for diagnosing HNSCC, for developing more effective, case-specific treatment, and for predicting survival. Using our extensive collection of tissue from HNSCC tumors and normal oral tissue of healthy controls, we will conduct the most comprehensive gene expression study of HNSCC to date, profiling the transcription levels of all human genes in an unprecedented number of HNSCCs. Building on recent findings from our group and others linking a subset of HNSCCs to oncogenic human papillomaviruses (HPV), we will assay HPV presence, genotypes, and gene expression profiles. This data-intensive analysis will form the basis for a larger epidemiologic evaluation of HNSCC to identify associations between gene expression signatures, the major risk factors for this cancer (tobacco, alcohol, HPV), and clinical outcomes. The specific aims are to: Aim 1: Use custom, virus-specific DNA microarrays to assay HNSCC tumors to detect HPV, HPV genotypes, HPV gene expression profiles, and whether HPV DNA is extra-chromosomal or chromosomally integrated in patterns associated with viral oncogene activation. Aim 2: Use DNA microarrays to profile the expression of all human genes in HPV-positive and -negative HNSCCs and healthy control tissue. Gene regulation up and down will be catalogued, clustered, and analyzed in association with Aim 1 data on HPV genotype, integration state and expression profile. Aim 3: Assess alterations in the cellular p53 and Rb tumor suppressor gene pathways in HPV-positive and HPVnegative tumors including p53, p14 ARF,and p16 lnk4agene structure, protein accumulation and function. Aim 4: Identify potential HNSCC biomarkers based on findings from aims 1-3 in combination with data on major HNSCC risk factors (tobacco, alcohol, HPV), and differences associated with staging and clinical outcomes. This project is a collaboration between individuals with expertise in molecular biology, genomics, bioinformatics, and epidemiology. Our joint studies of HNSCC will provide a rich output of data to support the goals of this project and to provide a foundation for future studies in basic oncogenesis, molecular epidemiology, and clinical issues of early diagnosis and treatment modalities.